Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS‐CoV‐2 Papain‐Like Protease

Abstract The two SARS‐CoV‐2 proteases, i. e. the main protease (Mpro) and the papain‐like protease (PLpro), which hydrolyze the viral polypeptide chain giving functional non‐structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high‐throughput mass spectrometry (MS)‐based assay which directly monitors PLpro catalysis in vitro. The assay was applied to investigate the effect of reported small‐molecule PLpro inhibitors and selected Mpro inhibitors on PLpro catalysis. The results reveal that some, but not all, PLpro inhibitor potencies differ substantially from those obtained using fluorescence‐based assays. Some substrate‐competing Mpro inhibitors, notably PF‐07321332 (nirmatrelvir) which is in clinical development, do not inhibit PLpro. Less selective Mpro inhibitors, e. g. auranofin, inhibit PLpro, highlighting the potential for dual PLpro/Mpro inhibition. MS‐based PLpro assays, which are orthogonal to widely employed fluorescence‐based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non‐covalent mechanisms.

(II) (a) Sequence and purification characteristics of the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2 used in this work. The consensus sequence (LXGG) for PL pro catalysis is in blue, the peptide cleavage site is in red; (b) mass spectrum (SPE-MS) of 2 (2.0 μM) in the reaction buffer (50 mM Tris, pH 8.0). m/z = 1034.03 corresponds to the +2 charge state of the nsp2/3 peptide 2; the enlarged region shows the major m/z +2 peak. m/z = 2067.04 corresponds to the +1 charge state of 2, m/z = 689.69 corresponds to the +3 charge state of 2, and m/z = 122.08 corresponds to the +1 charge state of Tris. The peptide is estimated to be >90% pure based on HPLC analysis. Note, m/z values referred to are for the most abundant isotope.
(III) (a) Sequence and purification characteristics of the SARS-CoV-2 nsp3/4 cleavage site-derived peptide 3 used in this work. The consensus sequence (LXGG) for PL pro catalysis is in blue, the peptide cleavage site is in red; (b) mass spectrum (SPE-MS) of 3 (2.0 μM) in the reaction buffer (50 mM Tris, pH 8.0). The m/z = 1041.13 corresponds to the +2 charge state of the nsp3/4 peptide 3; the enlarged region shows the m/z +2 peak. m/z = 694.42 corresponds to the +3 charge state of 3, m/z = 521.09 corresponds to the +4 charge state of 3, m/z = 417.06 corresponds to the +5 charge state of 3, and m/z = 122.08 corresponds to the +1 charge state of Tris. The peptide is estimated to be >90% pure based on HPLC analysis. Note, m/z values referred to are for the most abundant isotope.
(IV) (a) Sequence and purification characteristics of the N-acetylated N-terminal hydrolysis product (Ac-VTNNTFTLKGG) of the PL pro -catalyzed hydrolysis of the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2; this peptide was used as an internal standard in this work; (b) mass spectrum (SPE-MS) of the N-acetylated Nterminal hydrolysis product of 2 (2.0 μM) in the reaction buffer (50 mM Tris, pH 8.0). m/z = 1192.62 corresponds to the +1 charge state of the N-acetylated N-terminal hydrolysis product; the enlarged region shows the major m/z +1 peak. m/z = 1214.61 corresponds to the +1 charge state of the peptide as sodium adduct, m/z = 596.82 corresponds to the +2 charge state of the peptide, and m/z = 122.08 corresponds to the +1 charge state of Tris. The peptide is estimated to be >90% pure based on HPLC analysis. Note, m/z values referred to are for the most abundant isotope. Figure S2. SARS-CoV-2 PL pro endpoint assays reveal more efficient turnover of the SARS-CoV-2 nsp2/3 cleavage site-derived peptide than for the nsp1/2 or the nsp3/4 cleavage site-derived peptides. SARS-CoV-2 PL pro assays monitoring the hydrolysis of (a) the SARS-CoV-2 nsp1/2 (VTRELMRELNGG/AYTRYVDN, 1; Supporting Figure S1), (b) the SARS-CoV-2 nsp2/3 (VTNNTFTLKGG/APTKVTFGD, 2; Supporting Figure S1), and (c) the SARS-CoV-2 nsp3/4 (VVTTKIALKGG/KIVNNWLK, 3; Supporting Figure S1) cleavage site-derived peptides using SPE-MS reveal that the nsp2/3 peptide 2 is a more efficient substrate than the nsp1/2 1 and nsp3/4 3 peptides, in agreement with literature reports using related peptides. [2] Low levels of conversion were observed for the nsp3/4 peptide 3, whereas no turnover was observed under the reaction conditions for the nsp1/2 peptide 1. Conversion was observed to increase in a time-dependent manner, no peptide hydrolysis was detected in the absence of PL pro after incubation for 22 h (i.e. no enzyme controls). PL pro activity assays (50 μL total reaction volume) were performed using 0.2 μM PL pro and 2.0 μM substrate peptide in reaction buffer at 37 ⁰C in 0.5 mL Eppendorf tubes with agitation (300 rpm). The enzyme reactions were stopped after the indicated time by addition of 10%v/v aqueous formic acid (5 μL). The reaction mixtures were transferred into a 384 well plate (Greiner) and PL pro -catalyzed peptide cleavage was determined using SPE-MS as described in the Supporting Information (Section 4). An initial screen of buffer and reaction conditions revealed that PL pro catalysis was most efficient in Tris buffer (50 mM Tris, pH = 8.0) in the absence of any additives such as NaCl. While PL pro was active at ambient temperature (20 ⁰C), conversion was higher at 37 ⁰C. Note that conversions are estimates based on substrate depletion and thus not accurate, internal standards have not been used to quantify product formation. Figure S3. N-Terminal acetylated product peptides do not interfere with PL pro catalysis and can be used as internal standards to quantify PL pro turnover. The effect of the N-acetylated N-terminal and Cterminal product peptides of the PL pro -catalyzed hydrolysis of the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2 (VTNNTFTLKGG/APTKVTFGD, Supporting Figure S1) on PL pro catalysis was investigated using SPE-MS to investigate their utility as internal standards to quantify product formation. SPE-MS assays were performed in independent triplicates (n = 3; mean ± standard deviation, SD) as described in the Supporting Information (Section 4). Conditions: 0.2 μM PL pro , 2.0 μM of the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2, and 0.2 μM of the N-acetylated N-terminal product peptide (Ac-VTNNTFTLKGG; Supporting Figure   S1) and/or the N-acetylated C-terminal product peptide (Ac-APTKVTFGD; Supporting Figure S1) in buffer (50 mM Tris, pH 8.0, 20 °C). Note that the Ac-APTKVTFGD peptide is not a PL pro substrate; in the absence of the VTNNTFTLKGG/APTKVTFGD peptide in the reaction mixture, cleavage of the acetyl group from the Ac-APTKVTFGD peptide was not observed under the reaction conditions.

Supporting
(a) Peak areas of the extracted ion chromatograms (m/z = +2) for the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2 in the absence (black circles) and the presence (orange diamonds) of the Ac-VTNNTFTLKGG peptide.
The Ac-VTNNTFTLKGG peptide does not appear to affect PL pro catalysis and may thus be used as internal standard; (b) peak areas of the extracted ion chromatograms (m/z = +2) for the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2 in the absence (black circles) and the presence (lavender triangles) of the Ac-APTKVTFGD peptide. The Ac-APTKVTFGD peptide does not appear to affect PL pro catalysis and may thus be used as internal standard; (c) peak areas of the extracted ion chromatograms (m/z = +2) for the SARS-CoV-2 nsp2/3 cleavage sitederived peptide 2 in the absence (black circles) and the presence (green boxes) of both the Ac-VTNNTFTLKGG peptide and the Ac-APTKVTFGD peptide used as internal standards. The results reveal that PL pro -catalyzed cleavage of the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2 can be performed in the presence of the Ac-VTNNTFTLKGG peptide and the Ac-APTKVTFGD peptide without altering the reaction profile, thus enabling quantification of product formation by comparison of the integrals of the product peptide ion counts with those of the N-acetylated product peptides present in the reaction mixture as inert internal standards.
Supporting Figure S4. The SARS-CoV-2 nsp1/2 and nsp3/4-derived peptides do not affect the PL procatalyzed hydrolysis of the nsp2/3-derived peptide. The reaction profile of the PL pro -catalyzed hydrolysis of the SARS-CoV-2 nsp2/3-derived peptide 2 does not alter substantially in the presence of equimolar amounts of either the nsp1/2-derived peptide 1 (blue boxes) or the nsp3/4-derived peptide 3 (orange triangles) in the same reaction vessel, as revealed by comparison with a control reaction containing neither 1 or 3 (black inverse triangles).
Conversions of the SARS-CoV-2 nsp2/3-derived peptide 2 were determined by comparison of the peak areas of the extracted ion chromatograms (m/z = +2) for the N-terminal product peptide (VTNNTFTLKGG) with the one for the corresponding N-acetylated N-terminal product peptide, which was used as internal standard.

Supporting
Michaelis Menten kinetics of PL pro for the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2 afford a v max of 0.642 ± 0.0637 μM·s -1 and a K m of 49.4 ± 10.5 μM (goodness of fit: R 2 = 0.9496). Based on the assumption that the PL pro used was completely active, the k cat was calculated from the v max to be 3.21 ± 0.32 s -1 . k cat and K m are in the range of those reported for ISG15-Amc and K48 linked Ub2-Amc. [3] The specificity constant (k cat /K m ) was calculated to be 64980 ± 15256 M -1 ·s -1 , which is in the range of those reported using fluorescence-based PL pro assays as shown in Table 1. Figure S6. Robustness of the PL pro SPE-MS inhibition assays. SPE-MS inhibition assays were performed as described in the Supporting Information (Section 5). Z'-factors [4] for inhibition assay plates analyzed to determine IC50-values. The Z'-factors >0.5 (grey line) indicate a stable and robust assay of high quality. [4] Z'factors were determined according to the literature using Microsoft Excel. [4]

SARS-CoV-2 PL pro production and purification
The PL pro domain of nsp3 (region E746-T1063) was amplified from a synthetic fragment (Integrated DNA Technologies Ltd.) with In-Fusion adapted primers and cloned into a pOPINF derivative vector, containing an Nterminal hexa-histidine tag followed by a SUMO tag (ubiquitin like protein SMT3). The resultant plasmid was transformed into Lemo21(DE3) competent E. coli (NEB). Multiple transformant colonies were used to inoculate a starter culture supplemented with 0.5 mM rhamnose, 100 µg/mL carbenicillin and 34 µg/mL chloramphenicol.
The culture was then grown to log phase (200 rpm, 6 h, 37 °C). 10 mL of the starter culture was used to inoculate 1 L of auto induction medium (Formedium) supplemented with 10 mL of glycerol and 100 µg/mL carbenicillin. The cultures were grown at 200 rpm, 37 °C, for 5 h then switched to 18 °C for 72 h. The cells were harvested by centrifugation and stored at -80 °C. Approximately 40 g of cells were re-suspended in the purification buffer (50 mM Tris, pH 8, 300 mM NaCl) containing 10 mM imidazole and 0.03 μg/mL benzonase. Cells were lysed using an Avestin Emulsiflex homogeniser (3 passes, 30 kpsi, 4 °C). The lysate was centrifuged (50 000 g, 1 h, 4 °C). 10 mL of 50% His60 Ni Superflow Resin (Takara) was then added to the sample and stirred (1 h, 4 °C). The sample was applied to a gravity flow column, washed extensively using purification buffer containing 25 mM imidazole, and the protein was eluted using purification buffer containing 500 mM imidazole. Ubiquitin-like-specific protease 1 (Ulp1 protease) was added to the eluted protein at a ratio of 1:50 (w/w) to cleave the SUMO tag. The mixture was dialyzed against purification buffer containing 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) (overnight, 4 °C).
The protease and other impurities were removed from the cleaved target protein by reverse Nickel-NTA. PL pro was further purified by gel filtration chromatography using a S75 16/600 pg (Cytiva) column equilibrated in purification buffer. Relevant fractions were pooled, concentrated, flash frozen in aliquots and stored at -80 °C.
The enzyme was >95% pure as analyzed by SDS-PAGE and MS analysis and had the anticipated mass as reported; note that fresh aliquots, which were not frozen more than once, were used for inhibition assays.
The PL pro variants, i.e. H272K (nsp3 position H1017), D286A:Y268S (nsp3 D1031 and Y1013), Y268S (nsp3 Y1013) PL pro , were generated by overlap extension PCR and cloned by In-Fusion into the same pOPINF derivative vector as the wild type construct. The PL pro variants were produced and purified in a similar manner as described above.

Peptide synthesis
The PL pro substrate peptides mimicking the SARS-CoV-2 nsp1/2 (1), nsp2/3 (2), and nsp 3/4 (3) cleavage sites and the corresponding nsp2/3 (2)-derived N-acetylated C-terminal and N-terminal product peptides, which were used as internal standards, were prepared as C-terminal amides by solid phase peptide synthesis (SPPS) using commercially-sourced Fmoc-protected amino acids (Sigma-Aldrich, Inc.; Fluorochem Ltd.), peptide synthesis grade DMF (Sigma-Aldrich, Inc.), 20%v/v piperidine in DMF (AGTC Bioproducts Ltd.), HPLC-grade acetonitrile (Sigma-Aldrich, Inc), N,N-diisopropylcarbodiimide (Fluorochem Ltd.), oxyma (Sigma-Aldrich, Inc.) and Hünig's base (Sigma-Aldrich, Inc.). Microwave-assisted SPPS was performed using an automated peptide synthesizer (Liberty Blue, CEM Microwave Technology Ltd.) from the C-to N-terminus on Rink Amide MBHA resin (AGTC Bioproducts Ltd.; loading: 0.6-0.8 mmol/g) using iterative coupling (90 °C; 140 s; Fmoc-protected amino acids and N,N-diisopropylcarbodiimide, oxyma, Hünig's base) and deprotection steps (90 °C; 90 s; 20%v/v piperidine in DMF). For the synthesis of N-acetylated peptides to be used as internal standards, the free N-terminal amino group was capped after the final Fmoc-deprotection step using commercially-sourced N-acetoxysuccinimide (Tokyo Chemical Industry UK Ltd.) in DMF while the peptide was still immobilized on the resin. After washing the resin-bound peptides with dichloromethane, they were cleaved from the resin and simultaneously deprotected using a mixture of trifluoroacetic acid, triisopropylsilane, 1,3-dimethoxybenzene, and water (92.5/2.5/2.5/2.5%v/v, respectively). Solids were separated; the remaining clear solution was diluted with diethyl ether (45 mL/0.1 mmol resin). After incubation for 30 min at 0 °C, the mixture was centrifuged for 10 min using a Beckman Coulter Allegra X-30R centrifuge equipped with a SX4400 rotor (4500 rpm) and the supernatant discarded. The solid residue was dissolved in a water/acetonitrile mixture, frozen using liquid N2, and then lyophilized. The dried crude product was dissolved in a water/acetonitrile mixture, filtered, and purified using a semi-preparative HPLC machine (Shimadzu UK Ltd.) equipped with a reverse phase column (Gemini 00G-4454-U0-AX; phase: NX-C18). A linear gradient (typically 0−45%v/v over 35 min) of acetonitrile in MQ-grade water (each containing 0.1%v/v trifluoroacetic acid) was used as eluent. Fractions were analyzed by SPE-MS and those containing the pure peptide were combined and lyophilized. Sequences, mass spectra, and HPLC retention times for the peptides synthesized are shown in Supporting Figure S1.

PL pro SPE-MS assays
SARS-CoV-2 PL pro endpoint turnover assays were performed using 0.2 μM PL pro and 2.0 μM substrate peptide in reaction buffer at 20 ⁰C or 37 ⁰C in 0.5 mL Eppendorf tubes with agitation (300 rpm). The enzyme reactions were stopped by addition of 10%v/v aqueous formic acid (5 μL). The reaction mixtures were transferred into a 384-well polypropylene assay plate (Greiner) and analyzed by SPE-MS.
SARS-CoV-2 PL pro turnover assays for kinetic and competition experiments were performed in 96-well polypropylene assay plates (Greiner) with either a 1.0 or 0.5 mL final reaction volume; PL pro catalysis was directly monitored using SPE-MS. The RapidFire RF 365 high-throughput sampling robot used was programmed to aspirate samples from the reaction mixture at the indicated time intervals.
MS-analyses were performed using a RapidFire RF 365 high-throughput sampling robot (Agilent) attached to an iFunnel Agilent 6550 accurate mass quadrupole time-of-flight (Q-TOF) mass spectrometer operated in the positive ionization mode. Assay samples were aspirated under vacuum for 0.6 s and loaded onto a C4 solid phase extraction (SPE) cartridge. After loading, the C4 SPE cartridge was washed with 0.1%v/v aqueous formic acid to remove non-volatile buffer salts (5.5 s, 1.5 mL/min). The peptide was eluted from the SPE cartridge with 0.1%v/v aqueous formic acid in 85/15v/v acetonitrile/water into the mass spectrometer (5.5 s, 1.25 mL/min) and the SPE cartridge re-equilibrated with 0.1%v/v aqueous formic acid (0.5 s, 1.25 mL/min). The mass spectrometer parameters were: capillary voltage (4000 V), nozzle voltage (1000 V), fragmentor voltage (365 V), gas temperature (280 °C), gas flow (13 L/min), sheath gas temperature (350 °C), sheath gas flow (12 L/min). For data analysis, the m/z +2 charge states of the SARS-CoV-2 nsp1/2 (1), nsp2/3 (2), and nsp 3/4 (3) cleavage site-derived PL pro substrate peptides and/or the m/z +1 charge states of the corresponding product peptides were used to extract ion chromatogram data; peak areas were integrated using the RapidFire Integrator software (Agilent). To quantify product formation, the m/z +1 charge states of both the C-terminal and the N-terminal product peptides of the PL pro -catalyzed hydrolysis of the nsp2/3 peptide (2), as well as the corresponding Nacetylated C-terminal and N-terminal product peptides, which were used as internal standards, were used to extract ion chromatogram data; peak areas were integrated using the RapidFire Integrator software (Agilent). Data were exported into Microsoft Excel and used to calculate the product peptide concentrations using the equation: peptide concentration = 0.2 μM × (integral C-or N-terminal product peptide) / (integral N-acetylated C-or N-terminal product peptide).

PL pro inhibition assays
SPE-MS PL pro inhibition assays were performed using purified recombinant SARS-CoV-2 PL pro and the SARS-CoV-2 nsp2/3 cleavage site-derived peptide 2 (VTNNTFTLKGG/APTKVTFGD, Supporting Figure S1). Note that fresh PL pro aliquots, which were thawed and frozen not more than twice, were used for inhibition assays.